Despite the general in vitro susceptibility of Staphylococcus aureus isolates that cause infectious bovine mastitis, the pathogen remains difficult to eradicate with the available antibiotics. The capacity to survive intracellularly has been proposed as a factor contributing to the persistence of S. aureus in the bovine mammary gland. The costs associated with the use of cows or goats to assess the in vivo efficacy of new antibacterial compounds constitute a major drawback. Therefore, in the present study, a mouse model of intramammary infection has been characterized for in vivo testing of new experimental drugs. An inoculum of 100 CFU of S. aureus per gland caused an important level of infection with minimal tissue damage as observed at 24 h post-inoculation. By microscopy, polymorphonuclear neutrophil cell infiltration of the infected mammary glands was observed to increase over time. At 12-24 h of infection, the pathogen was primarily found alive and dividing in neutrophils and occasionally within mammary epithelial cells. Intramuscular or intravenous injections of cephapirin at t = 0 and 10 h reduced the number of CFU/g of gland in a dose-dependent manner. In conclusion, the mouse model of infectious mastitis proposed here is suitable for primary evaluation of experimental drugs after parenteral treatment of intramammary infection with a pathogen such as S. aureus that presents both intracellular and extracellular phases of growth.